Colorectal cancer is the second most common cause of new cancer cases and cancer deaths in the United States, with an estimated 146,940 new cases and 56,730 deaths in 2004 (Jemal, A. et al. (2004), Cancer statistics, 2004, CA Cancer J. Clin. 54:8-29).
Butyrate is a short-chain fatty-acid produced in the colon by bacterial fermentation of dietary fiber and required for colonic health. Tributyrin (glyceryl tributyrate) is a prodrug of butyrate that is hydrolyzed to butyrate in the intestine. It has been suggested as a therapeutic to prevent colon cancer (Conley, B. A., et al. (1998), Clin. Cancer Res. 4:629-634) and inflammatory bowel disease. Butyrate-producing bacteria have also been used to treat these conditions.
Short-chain fatty acids, including butyrate, are produced at high concentrations in the colonic lumen by bacterial fermentation of dietary fiber (Mortensen, P. B. & Clausen, M. R. “Short-chain fatty acids in the human colon: relation to gastrointestinal health and disease,” (1996) Scand. J. Gastroenterol. 216, 132-148; Manning, T. S. & Gibson, G. R. “Microbial-gut interactions in health and disease,” (2004) Prebiotics. Best Pract. Res. Clin. Gastroenterol. 18, 287-298). Of these, acetate is the most abundant, but butyrate plays the most important role in colonic physiology. In the proximal large bowel, butyrate represents the preferred respiratory fuel in the intestine through β-oxidation.
Apart from the function of butyrate as a dominant energy source for colonocytes, it also inhibits cellular proliferation and induces apoptosis by regulating the key proteins controlling the cell cycle (Coradini et al., (2000) Cell Prolif. 33(3):139-146). It induces differentiation in colon epithelial cells, but causes apoptosis in colon cancer cells (Gupta, N., Martin, P. M., Prasad, P. D. & Ganapathy, V. “SLC5A8 (SMCT1)-mediated transport of butyrate forms the basis for the tumor suppressive function of the transporter,” Life Sci. in press (2005)). The sodium salt of butyrate, sodium butyrate, is known to inhibit cell growth by favoring cell cycle arrest and promotes differentiation in normal as well as transformed cells (Barnard & Warwick, “Butyrate rapidly induces growth inhibition and differentiation in HT29 cells,” (1993) Cell Growth Differ., 4:495-501). Moreover, sodium butyrate induces apoptosis in a number of cancer cells (Mandal, M. and Kumar, R., “Bcl-2 expression regulates sodium butyrate-induced apoptosis in human MCF-7 breast cancer cells,” (1996) Cell Death Differ. 7:311-318; Bernhard, D. et al. “Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts,” (1999) FASEB J. 13:1991-2001; Giuliano, M. et al., “The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells,” (1999) Cancer Res. 59:5586-5595). Previous studies demonstrated that deficiency in the availability or utilization of butyrate causes colitis and may be involved in colon carcinogenesis (Soergel, K. H., (1994) Clin. Invest. 72:742-748).
Due to its growth-inhibiting and differentiation-inducing ability, butyrate was tested in the treatment of leukemia and solid tumors, together with analogues that have better pharmacodynamic properties, alone or in combination with other anti-cancer drugs (Miller et al., “Clinical pharmacology of sodium butyrate in patients with acute leukemia,” (1987) Euro J. Cancer Clin. Oncol. 23:1283-1287; Conley et al. “Phase I study of the orally administrated butyrate prodrug, tributyrin, in patients with solid tumors,” (1998) Clin Cancer Res. 4:629-634). Butyrate was also shown to induce WAF1/Cip1 (a potent inhibitor of cyclin-dependent kinases) mRNA in a human colorectal cancer cell line (WiDr), and cause G1-phase arrest (Katsunori N. et al., (1997) JBC 272(35):22199-22206). Due to its antiproliferative effects and lack of toxicity, butyrate has received attention as a potential cancer therapeutic agent.
G protein-coupled receptors are under intense scrutiny as potential targets of drug research, mostly because of the sheer size and diversity of this receptor family as well as the recognized high levels of specificity and sensitivity attainable by drugs targeting these receptors. Recently, Emmanuel et al. “Functional Characterization of Human Receptors for Short Chain Fatty Acids and Their Role in Polymorphonuclear Cell Activation,” (2003) J. Biol. Chem. 278(28):25481-25489, characterized two previously-designated orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Both butyrate and propionate are agonists for GPR41, whereas acetate was more selective for GPR43. The four genes encoding these receptors are intronless and are clustered onto chromosomal region 19q13.1. Although little information is available concerning these receptors, GPR41 was shown to induce apoptosis via the p53/Bax pathway in an ischemia/reperfusion paradigm (Kimura et al., (2001) J. Biol. Chem. 276(28):26453-26460).
Membrane transport in cells is a fundamental biological process that is mediated by various transporter and channel proteins. A major type of such proteins is a secondary active membrane transporter that uses a solute gradient to drive the translocation of other substrates (Mitchell, P., (1963) Biochem Soc. Symp. 22:141). Successful drug delivery will achieve an appropriate drug concentration at the target to elicit a desired level of response. Delivery of drugs through known transport systems has been under investigation for many years.
ATB0,+ is a broad substrate-specificity transporter that recognizes neutral as well as cationic amino acids as substrates. ATB0,+ is expressed primarily in the colon, lung and eye (Hatanaka, T. et al., (2003) J. Pharmacol. Exp. Ther. 308(3):1138-1147). ATB0,+ transports D-amino acids (Hatanaka, T. et al., (2002) Biochem. Biophys. Res. Commun. 291-295,), nitric acid synthase (NOS) inhibitors (Hatanaka, T. et al., (2001) J. Clin. Invest. 107(8):1035-1043,), and carnitine and its esters (Nakanishi et al., (2001) J. Physiol. 532(Pt 2):297-304).
SLC5A8 (SLC stands for solvent-linked carrier) was recently identified as a candidate tumor suppressor gene in humans that is silenced by methylation in colon cancer (Li, H, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 8412-8417). The protein encoded by SLC5A8 is a putative transporter belonging to the Na+/glucose cotransporter gene family. (Wright, E. M., and Turk, E. (2003) Pflugers Arch. Eur. J. Physiol. (Epub ahead of print, May 14, 2003)). SLC5A8 has been shown to transport Na+ when expressed in Xenopus oocytes (Li, H, et al. (2003), supra), but the cotransported organic/inorganic substrate has not been identified. Interestingly, the cloning of an identical cDNA has been reported independently by Rodriguez et al. (Rodriguez, A. M., et al. (2002) J. Clin. Endocrinol. Metab. 87, 3500-3503) who claimed that the cDNA codes for an uncoupled passive transporter for iodide. This reported functional feature of SLC5A8 as a passive iodide transporter has apparently led to the labeling of this transporter as SLC5A11 in a recent review by Wright and Turk (2003), supra). The findings by Li et al., (2003), supra that SLC5A8 is a Na+ transporter are in contradiction with those by Rodriguez et al., (2003), supra that the same protein functions as an uncoupled (i.e. no Na+ involvement in the transport process) iodide transporter.
SLC5A8 is a candidate tumor suppressor in human colon and silencing of its expression by epigenetic mechanisms represents an early event in the progression of colorectal cancer (Li, H, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 8412-8117). Re-expression of the gene in colon tumor cell lines prevents colony formation. This is the first time a plasma membrane transporter has been postulated to function as a tumor suppressor. SLC5A8 is a Na+-coupled transporter for short-chain fatty adds (acetate, propionate, and butyrate), lactate, pyruvate, and nicotinate (Miyauchi, S., Gopal, E., Fei, Y. J. & Ganapathy, V. “Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na+-coupled transporter for short-chain fatty acids,” J. Biol. Chem. 279, 13293-13296 (2004); Coady, M. J. et al. “The tumor suppressor gene SLC5A8 expresses a Na+-monocarboxylate cotransporter,” J. Physiol. (Lond.) 557, 719-731 (2004); Gopal, E. et al. “Expression of slc5a8 in kidney and its role in Na+-coupled transport of lactate,” J. Biol. Chem. 279, 44522-11532 (2004); Gopal, E. et al. “Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family,” Biochem. J. 388, 309-316 (2005)). Accordingly, SLC5A8 has been named SMCT1 (Sodium-coupled monocarboxylate transporter 1). As used herein, the terms SLC5A8 and SMCT1 are used interchangeably and refer to the same Na+-coupled transporter. It is not clearly known, however, how the transport function of SLC5A8 is related to its putative tumor suppressive role.
Pyruvate is the anionic form of the three-carbon organic acid, pyruvic acid. Pyruvate is a key intermediate in the glycolytic and pyruvate dehydrogenase pathways, which are involved in biological energy production. Pyruvate serves as a biological fuel by being converted to acetyl coenzyme A, which enters the tricarboxylic acid or Krebs cycle where it is metabolized to produce ATP aerobically. Energy can also be obtained anaerobically from pyruvate via its conversion to lactate. It has been suggested that 3-bromopyruvate may be effective as a cancer suppressor (Nelson, K. “3-Bromopyruvate kills cancer cells in animals,” (2002) Lancet. Oncol. 3(9):524; Geschwind et al. “Novel therapy for liver cancer: direct intraarterial injection of a potent inhibitor of ATP production,” (2002) Cancer Res 62(14):3909-13; Ko et al. “Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP,” (2004) Biochem. Biophys. Res. Commun. 324(1):269-75). As shown herein, the tumor suppressive role of SLC5A8 is associated with pyruvate-dependent inhibition of histone deacetylases (HDACs).
Butyrate is also an inhibitor of HDACs. HDAC inhibitors have shown promise in the treatment of cancer (Marks, P. et al. “Histone deacetylases and cancer: causes and therapies,” (2001) Nat. Rev. Cancer 1, 194-202; Vigushin, D. M. & Coombes, R. C. “Histone deacetylase inhibitors in cancer treatment,” (2002) Anti-cancer Drugs 13, 1-13; Davie, J. R. “Inhibition of histone deacetylase activity by butyrate,” (2003) J. Nutr. 133, 2485S-2493S; Drummond, D. C. et al., “Clinical development of histone deacetylase inhibitors as anticancer agents,” (2005) Annu. Rev. Pharmacol. Toxicol. 45, 495-528). The tumor-selective sensitization of cells to apoptosis by butyrate involves the tumor cell-specific induction of death receptor pathway or activation of the pro-apoptotic protein Bim (Nakata, S. et al. “Histone deacetylase inhibitors upregulate death receptor 5/TRAIL-R2 and sensitize apoptosis induced by TRAIL/APO2-L in human malignant tumor cells,” (2004) Oncogene 23, 6261-6271; Insinga, A. et al. “Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway,” (2005) Nat. Med. 11, 71-76; Nebbioso, A. et al. “Tumor-selective action of HDAC inhibitors involves TRAIL induction in acute myeloid leukemia cells,” (2005) Nat. Med. 11, 77-84; Zhao, Y. et al., “Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim,” (2005) Proc. Natl. Acad. Sci. USA 102, 16090-16095). Therefore, the entry of butyrate into colonic epithelial cells via SLC5A8 may explain the tumor suppressive role of the transporter in the colon.
As shown herein, SLC5A8 controls histone acetylation and apoptosis by mediating the entry of endogenous HDAC inhibitors, such as butyrate and pyruvate, into cells. Since SLC5A8, which transports butyrate from the colonic lumen into colonic epithelial cells, is downregulated in colon cancer, tributyrin, butyrate or similar tumor suppressors cannot be effectively targeted to colon cancer cells. Compositions and methods are needed for targeting biologically active molecules to cells where they are needed, especially under conditions in which the normal transport mechanisms for these molecules are impaired.